This application claims priority to International Patent Application PCT/AU98/00882 filed Oct. 23, 1998, and Australian Patent Application No. PP0120 filed Oct. 30, 1997.
This invention relates to methods and nucleic acid probes for assessing characteristics of lipid metabolism in animals, and in particular to methods of predicting fat levels in meat, milk, or other fat depots of animals. The invention is particularly applicable to predicting deposition of fat in muscular tissue, which produces the characteristic xe2x80x9cmarblingxe2x80x9d of meat, and to assessment of milk fat content. The methods of the invention are useful in selection of animals, particularly cattle, for ability to produce or high levels of marbling in meat, and to produce high or low levels of milk fat content.
The manner in which animals metabolise fat is of considerable economic significance in agriculture and animal husbandry. In some markets the high content of fat in meat, in the form of small fat deposits or xe2x80x9cmarblingxe2x80x9d, is regarded as highly desirable, and to induce heavy marbling of meat in cattle in particular the animals are grain fed for at least a short period prior to marketing and slaughter. In other markets a very lean meat is preferred. Similarly, a high fat content of milk is usually regarded as desirable. This can be particularly important if the milk is to be used for cheese production, and so these factors are important not only in cattle but also in sheep and goats. Recently generation of transgenic animals which secrete valuable proteins into their milk has been achieved, and in order to reduce the costs of purification of the desired protein a low content of fat in the milk is desirable.
Thus there is a need for methods by which the propensity of animals, particularly bovids and other ungulates, to deposit fat in muscle or to secrete fat into milk can be assessed.
Intramuscular or marbling fat is deposited in cattle between the fascicules of muscles, and usually develops when animals are fed a high calorie diet for a long time. The quantity of marbling fat is expressed either as a lipid concentration or as a standardised marbling score (eg. the Australian AUSMEAT standard). Unlike fat deposited in subcutaneous and renal depots, marbling fat is deposited continuously until relatively late in the development of the animal (Hood and Allen, 1973; Cianzio et al, 1985), and the amount of this fat is strongly correlated with the number of fat cells or adipocytes found in the muscle fascicules. Although some of the factors that are important in the differentiation of adipocytes are known (Ailhaud et al, 1992; Smas and Sul, 1995), the genetic factors that are involved in the difference between individuals in differentiation and development of the interfascicular adipocytes and deposition of fat were unknown, as were the genetic variants leading to a high or low marbling score.
To address this lack of information, we have obtained cattle samples from several breeds, the Angus, the Shorthorn and the Wagyu. These samples were readily differentiated due to their marbling score, with approximately half of the sample having a high marbling score and the other half of the sample having a low marbling score. We tested DNA markers from several regions of the bovine genome on the samples and the distribution of alleles was compared in the two groups.
Surprisingly, a significant association to marbling score was found with the anonymous DNA marker CSSM66. This marker had been assigned to bovine chromosome 14 (chr. 14) on the International Bovine Reference Family Panel (described in Barendse et al, 1997), with a location near the centromere. The gene for thyroglobulin (TG) is known to be located near this DNA marker (Barendse et al, 1997). TG is the molecular store for the thyroid hormones triiodothyronine and tetraiodothyronine, which have been implicated in the development of fat cells (Ailhaud et al, 1992; Darimont et al, 1993; Smas and Sul, 1995). TG has been sequenced in cattle (De Martynoff et al, 1987; Parma et al, 1987), and several DNA polymorphisms have been described previously (Georges et al, 1987). However, none of these polymorphisms is associated with fat or marbling.
We sought a polymorphism in the 5xe2x80x2 untranslated region (5xe2x80x2UTR) of TG in cattle, since the transcriptional and translational regulation of genes is mediated by the 5xe2x80x2UTR (Ptashne, 1988; Beato, 1989; Kozak, 1991).
A novel polymorphism in the 5xe2x80x2UTR of TG was identified, and shown to be correlated with marbling. This polymorphism can be used as a test to select animals for marbling performance, either as breeding stock or as animals to be fed for particular markets. Other characteristics of fat, such as fat thickness in other fat depots as well as fat percentage of tissues, including milk, are expected to be predicted by this marker, since the iodothyronines affect the general differentiation of adipocytes and since the influence of the level of the thyroid hormones on milk fat percentage is well known (Folley and Malpress, 1948). It is also expected that fat percentage of other mammalian species will be predicted by variation in the 5xe2x80x2UTR of the TG of those species.
In addition, we have surprisingly found significant associations between marbling score and the hitherto anonymous DNA markers CSSM34 and ETH10 on chromosome 5. CSSM34 is associated with retinoic acid receptor gamma (RARG), which is a known factor in the growth and differentiation of adipocytes. ETH10 is associated with retinol dehydrogenase 5 (RDH5), which catalyzes the interconversion of retinol and retinoic acid, and the level of retinol in the serum is directly related to intramuscular fat levels. The thyroid and steroid hormones such as thyroxine, retinol, and estrogen bind to a family of nuclear receptors with a similar set of hormone response elements. These nuclear receptors, such as RARG, initiate the transcription of genes, and are important elements in the growth, differentiation and specification of tissues. These elements are linked together structurally by similarities at the DNA sequence level.
In its general aspect the invention provides a method of assessing the fat metabolism characteristics of an animal, comprising the step of testing the animal for the presence or absence of one or more markers selected from the group consisting of:
a) an allele of the 5xe2x80x2 untranslated region of the gene encoding thyroglobulin;
b) an allele of the DNA polymorphism CSSM34, associated with the gene encoding retinoic acid receptor gamma (RARG); and
c) an allele of the DNA polymorphism ETH10, associated with 11-cis, 9-cis retinol dehydrogenase (RDH5).
According to a first embodiment the invention provides a method of assessing the fat metabolism characteristics of an animal, comprising the step of testing the animal for the presence or absence of an allele of the 5xe2x80x2 untranslated region of the gene encoding thyroglobulin.
Preferably the allele is allele 3, which indicates a high marbling score and/or high fat content of milk, or is allele 2, which indicates a low marbling score and/or low fat content in milk.
In a second embodiment the invention provides a method of identifying an animal with a high propensity for fat deposition in muscle (high marbling score), comprising the step of testing said animal for the presence or absence of allele 3 of the 5xe2x80x2 untranslated region of the gene encoding thyroglobulin, and selecting those animals possessing the allele. Preferably the animal is also tested for the presence or absence of allele 2 of the 5xe2x80x2 untranslated region of the gene encoding thyroglobulin, and those animals possessing allele 3 and not possessing allele 2 are selected. Most preferably the animal is homozygous for allele 3.
In a third embodiment, the invention provides a method of identifying an animal with a low propensity for fat deposition in muscle, comprising the step of testing the animal for the presence or absence of allele 2 of the 5xe2x80x2 untranslated region of the gene encoding thyroglobulin, and selecting those animals having allele 2. Preferably the animal is also tested for allele 3, and those animals having allele 2 but not allele 3 are selected. Most preferably the animal is homozygous for allele 2.
According to a fourth embodiment the invention provides a method of identifying an animal with a high propensity for fat deposition in muscle (high marbling score), comprising the step of testing the animal for the presence or absence of an allele of the DNA polymorphism CSSM34 associated with the gene encoding retinoic acid receptor gamma (RARG).
Preferably the allele is allele 2, which indicates a high marbling score. Preferably the animal is also tested for other alleles at the CSSM34 DNA polymorphism. For high marbling scores the animal is most preferably homozygous for allele 2. Allele 2 is 102 base pairs (bp) of DNA long.
According to a fifth embodiment the invention provides a method of identifying an animal with a low propensity for fat deposition in muscle, comprising the step of testing the animal for the presence or absence of an allele of the DNA polymorphism CSSM34 associated with the gene encoding retinoic acid receptor gamma.
Preferably the allele is allele 6, which indicates a low marbling score. Preferably the animal is also tested for other alleles at the CSSM34 DNA polymorphism. For low marbling scores the animal is most preferably homozygous for allele 6. Allele 6 is 112 bp of DNA long.
According to a sixth embodiment the invention provides a method of identifying an animal with intermediate propensity for fat deposition in muscle (low marbling score), comprising the step of testing the animal for the presence or absence of an allele of the DNA polymorphism CSSM34 associated with the gene retinoic acid receptor gamma.
Preferably the allele is one or more of alleles 1, 3, 4, and 5 which indicates an intermediate marbling score. Preferably the animal is also tested for other alleles at the CSSM34 DNA polymorphism. The sizes of the alleles are given in Table 11. There is no special preference for genotype with these alleles. Other alleles may occur at CSSM34 with different lengths of DNA.
In a seventh embodiment, the invention provides a method of identifying an animal of, or derived from, the Wagyu cattle breed with a high propensity for fat deposition in muscle, comprising the step of testing the animal for the presence or absence of an allele of the ETH10 DNA marker. Preferably the allele is allele 5. Allele 5 is 223 bp long.
In an eighth embodiment, the invention provides a method of identifying an animal of, or derived from, the Wagyu cattle breed with a low propensity for fat deposition in muscle, comprising the step of testing the animal for the presence or absence of an allele of the ETH10 DNA marker. Preferably the allele is allele 2. Allele 2 is 217 bp long.
These embodiments of the invention are also applicable to the selection of animals for high or low fat content of milk respectively. The method is also useful for testing for fat levels in carcases.
According to a second aspect the invention provides a method of detecting one or more of the alleles of the invention in an animal, comprising the steps of:
a) obtaining a biological sample from the animal,
b) extracting DNA from the sample,
c) amplifying DNA from the relevant gene, and
d) identifying alleles in the amplified DNA.
Preferably the DNA is either of the 5xe2x80x2 untranslated region of thyroglobulin or of DNA segments near the retinoic acid receptor gamma; if the animal is of the Wagyu breed of cattle, the DNA segments are near the retinol dehydrogenase 5 gene.
Preferably the biological sample is blood, but other biological samples from which DNA can be amplified may be used. For example hair root samples, cheek scrapings, skin samples and the like may be used. Preferably for alleles of the 5xe2x80x2 untranslated region of the thyroglobulin gene the region of DNA amplified includes a homopurine sequence and a copy of the monomeric dispersed repeat sequence. Preferably amplification is performed using polymerase chain reaction, but other DNA amplification methods such as ligase chain reaction are well known in the art, and may alternatively be used. Preferably the alleles are identified by polyacrylamide gel electrophoresis.
In a third aspect the invention provides oligonucleotide probes for amplification of the markers of the invention, selected from the group consisting of:
a) oligonucleotide probes for the 5xe2x80x2 untranslated region of the thyroglobulin gene, having the sequences
b) oligonucleotide probes for amplication of the CSSM34 DNA marker, with the sequences
c) oligonucleotide probes for amplication of fragments from the RARG gene in cattle, with sequences
d) oligonucleotide probes for amplification of fragments from the RDH5 gene in cattle, with sequences
e) oligonucleotide probes for amplification of the ETH10 marker in Wagyu cattle, with sequences:
In a fourth aspect the invention identifies Yeast Artificial Chromosomes, which are positive by hybridization to the oligonucleotide primers for CSSM34U and CSSM34D as well as for RARGE8U2 and RARGE8D1. These are 77D3, 77E3, 71G8, 94B4 and 71E4.
In a sixth aspect the invention provides an isolated nucleic acid molecule encoding part of the bovine retinoic acid receptor gamma, having the sequence set out in SEQ ID NO: 8 as defined herein.
The methods of the invention may be used both for the selection of breeding animals and for the selection of unpedigreed animals for entry into feed lots. In the latter case, the methods of the invention are applicable to deciding the length of time which animals spend in feedlots, since a high marbling score is unlikely to be attained with animals which are homozygous for allele 2 of the 5xe2x80x2 untranslated region of thyroglobulin or allele 6 of CSSM34, or a Wagyu animal with allele 2 of ETH10, even after long feedlot holding.
The methods of the invention are applicable to animals including but not limited to cattle and other bovids, including water buffalo and bison, to other ungulates, including sheep, goats and deer, and to pigs.
For the purposes of this specification it will be clearly understood that the word xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluding but not limited toxe2x80x9d, and that the word xe2x80x9ccomprisesxe2x80x9d has a corresponding meaning.